During the currently funded study, prostaglandin E2 (PGE2) was shown to stimulate invasion and metastasis by metastatic Lewis lung carcinoma (LLC) clones. This appeared to be dependent on generation of a cAMP response and, in turn, activation of protein kinase A (PKA). In nonmetastatic LLC, PGE2 elevated cAMP levels, but this did not significantly activate PKA or stimulate metastatic properties, even though their PKA could be activated extracellularly. These data suggest that PKA activity and PKA-stimulated metastasis may be more tightly regulated in nonmetastatic LLC than in metastatic LLC. Further studies implicated dephosphorylation reactions of protein phosphatases (PP) -1 and -2A as the regulatory controls for PKA in nonmetastatic LLC. The hypothesis of this study is that in metastatic LLC, defective regulation of PKA activity by PP-1/2A leads to a heightened PKA response to PGE2 and, in turn, stimulation of metastasis. Studies to assess the role of PKA activation in stimulation of metastasis will be accomplished with the use of wild-type metastatic and nonmetastatic LLC, LLC transfectants that either overexpress the Calpha subunit of PKA or express a mutant RIalpha that blocks PKA activation, and by modulating the level of expression of these subunits with Zn2+. The first objective of this study will identify if and how PKA activation in LLC variants leads to increased metastasis from a s.c. or orthotopically implanted tumor site. The second objective will identify points at which metastatic and nonmetastatis LLC might diverge in the PKA activation process. The third objective will determine if the balance between phosphorylation by PKA and dephosphorylation by PP-1/2A regulates the metastatic properties of LLC. These studies will result in an understanding of intracellular mechanisms which regulate PKA activity and how defects in these regulatory controls increase metastasis. Future studies will then be planned to develop strategies by which to stimulate PP-1/2A so as to block PKA stimulation of metastasis.